1. Field of the Invention
The present invention relates to the field of plant breeding, specifically to methods of soybean breeding and the resulting soybean plants and soybean lines. More particularly, the invention relates to soybean cyst nematode-resistant soybean lines and methods of breeding same, the methods involving molecular marker analysis.
2. Discussion of Related Art
Soybeans are a major cash crop and investment commodity in North America and elsewhere. Soybean oil is one of the most widely used edible oils, and soybeans are used worldwide both in animal feed and in human food production. The soybean cake, or meal, that remains after processing the beans for oil, is a high-protein foodstuff used extensively in livestock and poultry rations. It is an excellent protein with respect to most of the essential amino acids and also a good source of vitamins of the B-complex.
Nematodes are small wormlike animals, many of which are plant, animal or human parasites which cause a variety of diseases. Plant pathogenic nematodes are a major agricultural problem causing significant crop and yield losses. Plant tissue, particularly root tissue, is damaged by nematode feeding. Such feeding can cause mechanical tissue damage and the accompanying injection of nematode enzymes can cause further tissue disintegration. Nematode infections of roots result in root galls, and distortions in root growth. Similar symptoms accompany nematode infections of other parts of the plants.
Nematode infection can also be accompanied by bacterial or fungal infection. In such plant-disease complexes, damage caused by nematodes can lead to enhanced severity of bacterial or fungal infection. In addition, several nematodes are vectors for plant pathogenic viruses.
The soybean cyst nematode, Heteroderaglycines, was apparently first identified on soybeans in the United States in 1954 at Castle Hayne, N.C. Since its discovery the soybean cyst nematode ("SCN") has been recognized as one of the most destructive pests in soybean. It has been reported in nearly all states in which soybeans are grown, and it causes major production problems in several states, being particularly destructive in the Midwestern states.
Although the use of nematocides is effective in reducing the population level of the nematode, nematocide use is both uneconomical and potentially environmentally unsound as a control measure in soybean production. Neither is crop rotation a practical means of nematode control, because rotation with a SCN-resistant crop for at least two years is necessary for reducing soybean losses and there currently exist no known SCN-resistant soybean plant lines which are commercially satisfactory. It has long been felt by soybean breeders that use of commercially satisfactory SCN-resistant varieties would be the most practical control measure. Therefore, there exists a great need in the relevant art for soybean plants and plant lines which are commercially satisfactory and are resistant to soybean cyst nematodes.
Screening of soybean germplasm for resistance to SCNs was begun soon after the discovery of the nematode in the United States. Although various soybean lines have proven resistant to various races of soybean cyst nematode, the plant introduction PI437654 is the only soybean line which has been shown to have resistance to all known SCN races (3, 1, 2, 5, 14, 6 and 9). However, a disadvantage of PI437654 is that its physical characteristics include a black seed coat, poor standability, seed shattering, and low yield, making it commercially unsatisfactory and making desirable the introgression of its SCN resistance into elite germplasm with a minimum of linkage drag. Conventional breeding with PI437654 produced the variety "Hartwig", which is more adapted to cultivation and can be used as a source of SCN resistance in soybean breeding; however, Hartwig retains and transfers the above-mentioned poor physical characteristics. Prior to the present invention, soybean breeders have been unable to selectively introgress SCN resistance without these poor physical characteristics.
Resistance to SCN has been shown to be multigenic and quantitative in soybean, although complete resistance can be scored qualitatively. It has previously been estimated that PI437654 has three genes required for complete resistance to race-3, four genes for race-5, and three genes for race-14. The multiple genes and SCN races involved contribute to the difficulty breeders have in developing soybean varieties having SCN resistance.
When considering the development of improved plant lines, a great deal of emphasis is usually placed on the strategy of introducing characteristics into plants via genetic engineering techniques. While there is excitement over advances in plant genetic engineering, the prospects for the general use of these techniques for plant improvement are tempered by the realization that very few genes corresponding to plant traits of interest have been identified. The use of direct gene transfer in manipulating these traits, of course, is therefore difficult due to problems in pinpointing and then cloning those individual loci which contribute predominantly to the expression of the trait.
Alternatively, much attention is being given to selective breeding techniques for introgressing one or more desired traits from one soybean plant line into another plant line having other desired traits. A procedure that has been used by plant breeders to increase efficiency in the testing of traits which are difficult or expensive to evaluate is the use of indirect selection criteria. One indirect selection criterion, for example, might be an easily recognized morphological characteristic of the plant which is either genetically linked to the desired trait or perhaps a component of the desired trait, e.g., the association between leaf size and seed size in beans.
Agronomically important traits such as, for example, plant yield, height, maturity, fruit and grain characteristics, and nematode resistance are all attractive targets for manipulation in plant improvement programs, but these traits often have very low heritabilities. Heritability is the proportion of observed variation in a particular trait that can be attributed to inherited genetic factors in contrast to environmental ones and, therefore, is important to the efficiency of the selection process. Influencing heritability of such traits, sometimes termed "quantitative" traits, is difficult, however, because expression of a number of different gene products generally influences the phenotype. Quantitative traits are thus often characterized by continuous rather than discreet distribution of phenotypic expression. There is currently a poor understanding of how single genes influence the expression of complex traits and, in conventional plant breeding programs, selection for inheritance of quantitative traits is difficult due to the unrecognized genetic basis of the trait. Determination of genotypic information from phenotypic values is further imprecise because evaluation of the trait may frequently be confounded by environmental effects.
A method of introgressing multigenic quantitative traits into wild germ plasm has been described by which the role of individual plant genes in quantitative trait expression may be identified and characterized. This method involves the determination of genetic markers closely linked to important genes, and the indirect selection for favorable alleles based upon the presence of the specific markers. This method allows selection to be accomplished more efficiently than direct phenotypic selection.
A class of plant molecular markers which has gained widespread acceptance is based upon restriction fragment length polymorphisms ("RFLP"s). Generally, RFLPs are differences observed between genotypes in the fragment lengths of restriction endonuclease-digested DNA. RFLPs occur as a result of base pair or positional changes in the restriction enzyme recognition sites which flank a chromosomal location and can be detected by hybridization of labelled DNA clones containing sequences that are homologous to a portion of the chromosomal fragment. Hybridization with a unique cloned sequence can permit the identification of a specific chromosomal region, or locus.
This technology conventionally employs cloned DNA fragments to detect differences between individuals at the DNA sequence level. When genomic DNAs from two genetically distinct individuals are digested with a restriction enzyme, electrophoresed and probed with a labelled DNA clone, polymorphisms in the hybridization patterns sometimes result due to sequence differences between the individuals. The term "restriction fragment length polymorphism" has been coined to describe this variation.
Differences in fragment lengths which are revealed, for example, by agarose gel electrophoresis, function as alleles of the RFLP. Thus, RFLPs can serve as genetic markers in a manner analogous to conventional morphological or isozyme markers. Unlike most genetic markers, however, they are not the products of transcription and translation. Additionally, RFLPs possess certain additional advantages over previously available genetic markers. First, RFLPs reflect existing differences between genetically distinct individuals. The potential number of RFLPs for all practical purposes is thus unlimited, as digestion of the genomic DNA of any higher eukaryote with a six base recognition enzyme will generate more than a million fragments, many of which can be polymorphic. Additionally, over one hundred different restriction enzymes have now been described, each of which may generate a new and different set of fragments.
RFLP markers rarely possess detectable phenotype effects of their own, so they can be utilized in economic lines without detriment and many can be evaluated at one time without the pleiotropic effects often seen with phenotypic markers. Evaluation can be performed on small amounts of DNA obtained from plant tissue at virtually any stage of plant development from seeds, to roots, to shoots, to fruits, or even with tissue culture material. Evaluation of RFLPs is not affected by environmental factors and greenhouse-grown plants will not differ from field-grown plants when tested. Finally, the evaluation of RFLPs reveals the exact genotype, so the heterozygous state can be differentiated from the homozygous condition at any chromosomal location.
Numerous direct applications of RFLP technology to facilitate plant breeding programs have been suggested. Because of the large numbers of RFLP markers available in a population of interest, one of the more important applications of RFLPs is as markers linked to genes affecting the expression of quantitatively inherited traits. A prerequisite for the use of RFLPs as indirect selection criteria is the identification of RFLPs closely linked to the genomic loci affecting expression of the trait of interest. Such genomic loci are commonly referred to as quantitative trait loci ("QTL"s).
The introgression of quantitative traits from one germplasm to another conventionally involves the identification of favorable genotypes in a segregating generation followed by repeated backcrossing to commercially acceptable cultivars. This procedure is feasible for simply inherited quantitative traits, but as the number of genes controlling a trait increases, screening the number of F2 segregants required to identify at least one individual which represents the ideal (homozygous) genotype quickly becomes prohibitive. For example, with one gene and two alleles of equal frequency, the probability of recovering a desirable genotype on the F2 generation is 1/4. However, if the number of genes is increased to 5 or 10, the probability of recovering an ideal genotype in the F2 population is reduced to approximately one in one thousand and one in one million, respectively. Thus, to identify desirable segregants, one must either reduce the number of segregants needed or have available very efficient screening procedures.
One described method of RFLP research involves crossing a plant source (designated P.sub.1) having a desired multigenic trait, for example, SCN resistance, with a second plant (designated P.sub.2) having essentially or substantially opposite characteristics, that is, SCN susceptibility. Heterozygous plants from the F1 population are selfed to create a segregating (F2) plant population which exhibits a gradient with respect to the degree of expression of the multigenic or quantitative trait of interest, e.g., SCN resistance.
Quantitative values for the trait of interest (SCN resistance) are determined and assigned to each individual parent plant, F1 population plant, and F2 segregating plant and a genomic DNA sample from each plant is prepared for Southern blotting. Following preparation for a Southern blot, an RFLP probe is randomly chosen or selected from an RFLP genetic linkage map and hybridized to create the blot. Additional Southern blots are constructed using other RFLP probes, and the degree of association between the trait of interest and each particular RFLP is determined. Additionally, in a multigenic system such as SCN resistance, the relative importance of each correlating RFLP can be determined. Particular values can be assigned to those RFLPs and utilized in a mathematical model to assist in predicting the degree of trait expression in a particular plant. In this manner, the RFLP marker(s) having the strongest association with the trait of interest can be determined and utilized, for example, in a breeding program to select plants having SCN resistance.
It is of particular importance, both to the soybean breeder and to farmers who grow and sell soybeans as a cash crop, to identify, through genetic mapping, QTLs associated with resistance to the various SCN races and to identify markers associated thereto which may be used to introgress SCN resistance with a minimal amount of linkage drag. Knowing these superior markers, soybean breeders will be better able to breed SCN resistant soybeans which also possess the other genotypic and phenotypic characteristics desired for commercial soybean plant lines. Superior markers and plants and plant lines developed using the same are provided by the present invention. Also provided are improved methods for identifying molecular markers linked to SCN resistance QTLs.